Abstract Background The advent of therapeutic monoclonal antibodies (t-mAbs) has greatly improved the prognosis of patients with multiple myeloma and other monoclonal gammopathies. However, the standard laboratory tests used to diagnose and monitor these conditions are unable to distinguish between t-mAbs and the M-protein produced by the patient’s clonal plasma cells. Mass spectrometry-based methods for detecting and quantifying M-proteins provide a potential solution to this problem because they are based on a protein’s mass-to-charge ratio (m/z) rather than on charge alone. Since each antibody has a unique amino acid composition and therefore a unique mass, most antibodies can be distinguished from each other via mass spectrometry, even if they have the same heavy and light chain isotypes. The EXENT System (The Binding Site, part of Thermo Fisher Scientific) combines automated immunoprecipitation of immunoglobulins with MALDI-TOF mass spectrometry for the detection, quantitation, and isotyping of M-proteins. This technology has the potential to become part of the diagnostic and monitoring algorithm in the workup for monoclonal gammopathies. The purpose of this study was to assess the reproducibility of the EXENT System in measuring the m/z values of 6 commonly encountered t-mAbs and to compare measured m/z values to calculated expected values for each t-mAb. Methods Daratumumab, elotuzumab, isatuximab-irfc, rituximab-abbs, pembrolizumab, and nivolumab were obtained from the hospital pharmacy and diluted to a concentration of 1 mg/mL in normal human serum. Imprecision of t-mAb m/z values was assessed by measuring 5 aliquots of each t-mAb on the EXENT System daily for a total of 5 days. Intra-day and inter-day mean, standard deviation (SD), and coefficient of variation were calculated for the m/z values of the +2 charge state for each t-mAb light chain. Inter-day mean m/z values were compared to expected m/z values for each t-mAb, which were calculated using the IMGT t-mAb protein database and the Expasy Compute pI/MW tool. Results The inter-day mean m/z values of the +2 charge state for each t-mAb were as follows: 11,690.4 for daratumumab, 11,712.5 for elotuzumab, 11,744.1 for isatuximab, 11,519.1 for rituximab, 11,871.5 for pembrolizumab, and 11,685.5 for nivolumab. Measurements were highly precise, with both intra-day and inter-day SDs consistently lower than +/- 0.8 m/z. With the exception of daratumumab and rituximab, the measured t-mAb inter-day mean m/z values were approximately 2.0 m/z lower than the expected values. For daratumumab, the measured value was 0.6 m/z lower than the expected value; for rituximab, the measured value was 0.7 m/z higher than the expected value. Conclusion The EXENT System measured the m/z of the +2 charge state of each t-mAb with excellent intra- and inter-day precision. The difference of 2.0 m/z observed between measured and expected values for 4 of the 6 t-mAbs tested suggests that laboratories should perform their own imprecision studies when setting up monoclonal gammopathy testing on the EXENT System and set interferent flags accordingly. In our case, we used inter-day mean m/z values to implement interferent flags for each t-mAb with a mass tolerance of +/- 2.0.
Villar et al. (Wed,) studied this question.